This invention relates to molecular gene switches that use molecules capable of binding a specific DNA sequence in a ligand-dependent manner where the ligand itself is capable of binding DNA. Moreover, this invention relates to methods for the identification of said ligand-dependent DNA binding molecules.
Gene switches are currently of great interest to those wishing to control timing and/or dosage of gene expression. Various gene switches have been developed in the prior art. Most of these prior art switches are derived from gene regulatory proteins. In these systems, the switching ligand binds to the protein, inducing a protein conformational change that affects DNA binding.
It is often the case that a gene""s expression is affected by one or more different protein(s). Diverse proteins may influence expression of the same gene. Said protein(s) may be present in a first cell or cell type, but these protein(s) may be absent from a second cell or cell type. Therefore, a molecule which affects only a single known regulatory protein will not have any effect on the expression of the same gene in a cell where this particular regulatory protein is not expressed, or is otherwise sequestered. Thus, one of the difficulties of the prior art is that a protein-binding switching molecule will have no effect on the expression of a gene if the particular protein to which the switching molecule binds is not present.
Similarly, a gene""s expression may be affected by numerous different proteins in different cells or cell types. A molecule which affects only a single known regulatory protein will not have any effect on the expression of the same gene in a cell in which its expression is controlled by a different protein or proteins. Therefore, one of the difficulties in the prior art is that a plurality of switching molecules may be required in order to modulate or switch the expression of a single gene.
Therefore, in order to effect switching of gene expression at a given DNA sequence, independently of the particular activator protein, it is desirable to target the DNA. Further, custom DNA binding proteins would benefit from switches; if these could be designed to interact with DNA, there would be a greater freedom in the design of said proteins.
There are numerous polypeptide modifications which are known to affect their interaction with a broad spectrum of molecules such as nucleic acids, polypeptides (both intra- and inter-molecularly) other macromolecular structures such as membranes, small molecules, ions, or other entities. Clearly, it is a problem that polypeptide modifications may compromise the binding of prior art switching molecules to their polypeptide targets.
The present invention seeks to overcome such difficulties.
Aspects of the present invention are set out in the claims and are described below.
In a first aspect, the present invention provides a method of selecting a gene switch, which gene switch comprises (i) a target DNA molecule; (ii) a DNA binding molecule which binds to the target DNA molecule in a manner modulatable by a DNA binding ligand; and (iii) the DNA binding ligand, which method comprises:
(a) contacting one or more candidate target DNA molecule(s) with one or more candidate DNA binding molecules, in the presence of one or more DNA binding ligands, wherein at least one of the candidate DNA binding molecules comprises a non-naturally occurring DNA binding domain;
(b) selecting a complex comprising a candidate target DNA, a DNA binding molecule and a DNA binding ligand;
(c) isolating and/or identifying the unknown components of the complex;
(d) comparing the binding of the DNA binding molecule component of the complex to the target DNA component of the complex in the presence and absence of the DNA binding ligand component of the complex; and
(e) selecting complexes where said binding differs in the presence and absence of the DNA binding ligand component.
Preferably the DNA binding molecules are provided as a plurality of DNA binding molecules, more preferably as a library of DNA binding molecules. Where only one DNA binding molecule is included in the screen, the DNA binding molecule comprises a non-naturally occurring DNA binding domain. The term xe2x80x9ca non-naturally occurring DNA binding domainxe2x80x9d means that the DNA binding domain does not occur in nature, even as part of a larger molecule, and has been obtained by deliberate mutagensis procedures or de novo design techniques.
Preferably the target DNA is provided as a plurality of DNA sequences, more preferably as a library of DNA sequences, said sequences being related to one another by sequence homology.
In one embodiment, a plurality of candidate DNA binding ligands are used, in which case is preferred to use one target DNA.
Typically one of the components isolated and/or identified in step (c) is a DNA binding ligand component or a DNA binding molecule component.
In a preferred embodiment of the first aspect of the invention, the selected DNA binding molecule component has a higher affinity for the target DNA in the presence of the DNA binding ligand component than in the absence of the DNA binding ligand component.
Alternatively, the selected DNA binding molecule component has a higher affinity for the target DNA in the absence of the DNA binding ligand component than in the presence of the DNA binding ligand component.
In a highly preferred embodiment, the candidate DNA binding molecules are provided as a phage display library.
The method of the present invention may be used to select a DNA binding molecule which binds to a target DNA molecule in a manner modulatable by a DNA binding ligand.
The method of the present invention may also be used to select a target DNA to which binds a DNA binding molecule in a manner modulatable by a DNA binding ligand.
The method of the present invention may also further be used to select a DNA binding ligand that modulates binding of a DNA binding molecule to a target DNA.
Generally, the DNA binding ligand and the DNA binding molecule are different
In a preferred aspect of the invention, said candidate molecules are polypeptides. In a more preferred embodiment, said candidate molecules are polypeptides at least partly derived from transcription factors. In an even more preferred embodiment, said candidate molecules are derived from zinc finger transcription factors.
Advantageously, the candidate DNA binding molecules are provided as a phage display library.
In a preferred aspect of the invention, the DNA binding ligand is selected from Distamycin A, Actinomycin D and echinomycin.
In another aspect, the invention relates a gene switch comprising (i) a target DNA molecule; (ii) a DNA binding molecule which binds to the target DNA molecule in a manner modulatable by a DNA binding ligand; and (iii) the DNA binding ligand. In particular, the present invention relates to DNA binding molecules and/or DNA binding ligands and/or target DNA obtainable by the methods disclosed herein.
The present invention also provides a method for engineering a novel class of gene switches in which a DNA binding ligand affects or modulates the interaction of a DNA binding molecule (for example phage displayed polypeptide), with its target DNA. In a preferred aspect, the present invention relates to the selection of DNA binding polypeptides which recognise a particular DNA sequence or structure. Preferably, said method may include selection of phage displayed polypeptides that bind a DNA target in the presence or absence of one or more DNA binding ligands. Of the phage displayed polypeptides which are selected under these conditions, some may bind the DNA with higher affinity in the presence of ligand. whereas others may bind the DNA with higher affinity in the absence of ligand.
The gene switches and components thereof can be used in methods of regulating gene expression. Accordingly, the present invention also provides a method of modulating the expression of one or more genes, said method comprising administering a DNA binding molecule and DNA binding ligand selected according to the method of the invention to a cell wherein the regulatory sequences of said genes comprise a target DNA selected according to the method of the invention.
The present invention also provides a method of modulating the expression of one or more nucleotide sequences of interest in a host cell which host cell comprises a nucleic acid sequence capable of directing the expression of a DNA binding molecule and a target DNA sequence to which the DNA binding molecule binds in a manner modulatable by a DNA binding ligand which method comprises administering said DNA binding ligand to the cell and wherein the DNA binding molecule is heterologous to the host cell.
Preferably the host cell is a plant cell. More preferably the plant cell is part of a plant and the target sequence is part of a regulatory sequence to which the nucleotide sequence of interest is operably linked, said regulatory sequence being preferentially active in the male or female organs of the plant.
In a further aspect there is provided the use of a DNA binding molecule selected by the method of the invention in a method of regulating transcription from a DNA sequence comprising a target DNA to which the DNA binding molecule binds in a manner modulatable by a DNA binding ligand.
Also provided is the use of a DNA binding ligand selected by the method of the invention in a method of regulating transcription from a DNA sequence comprising a target DNA to which a DNA binding molecule binds in a manner modulatable by the DNA binding ligand.
Also provided is the use of a target DNA selected by the method of the invention in a method of regulating transcription from a DNA sequence comprising the target DNA to which a DNA binding molecule binds in a manner modulatable by a DNA binding ligand.
In another aspect, the present invention provides a non human transgenic organism comprising a target DNA sequence and a nucleic acid sequence capable of directing the expression of a DNA binding molecule which binds to the target DNA in a manner modulatable by a DNA binding ligand wherein the target DNA sequence and/or nucleic acid sequence are heterologous to the organism.
Preferably the transgenic non-human organism is a plant.